Micro-fluidic system using micro-apertures for high throughput detection of cells

ABSTRACT

A microfluidic detection system for micrometer-sized entities, such as biological cells, includes a detector component incorporating a plate with a plurality of opening, the plate separating two chambers, one in communication with a fluid source containing target entities bound to magnetic beads. The openings are sized to always permit passage of the magnetic beads therethrough into a lower one of the chambers and are further sized to always prevent passage of the target entities from the upper one of the chambers. The detector component further includes a magnet positioned to pull unbound magnetic beads through the openings and to capture target entities bound to magnetic beads on the surface of the plate. In a further feature, the microfluidic detection system is configured to pass target molecules through the plate to be bound to a functionalized surface of the lower chamber.

PRIORITY CLAIM

The present application is a division of Ser. No. 14/037,476, filed onSep. 26, 2013, which is a continuation-in-part of co-pending applicationSer. No. 14/001,963, filed on Aug. 28, 2013, which is a national stageunder 35 U.S.C. § 371 of international application PCT/US2012/032356,filed on Apr. 5, 2012, which claims priority to provisional applicationSer. No. 61/471,762, filed on Apr. 5, 2011, in the name of the sameinventors, the entire disclosure of which is incorporated herein byreference.

TECHNICAL FIELD

The present invention generally relates to microfluidics andparticularly to detecting targeted entities, such as cells andmolecules, present in sample fluids.

BACKGROUND

Circulating tumor cells (or CTCs) are rare cells present in the blood ofmetastatic cancer patients. Quantitative detection of CTCs is importantfor early detection of cancer as well as monitoring of the diseaseprogression and response to therapy. CTC count correlates with overalltumor burden and can often serve as more reliable indicators ofmetastatic disease than molecular disease markers. For example, thelevel of prostate specific antigen (PSA) can often rise due to benignprostate hyperplasia (common in people over 60) and hence may notnecessarily indicate cancer.

The presence of a significant number of CTCs can be a reliable indicatorof the presence of cancer. Also, possible recurrence after surgery canbe detected much earlier by CTCs than by most molecular markers. Anotheradvantage is that CTCs can be further interrogated after detection;sequencing of the genome and transcriptome could reveal the mutationsthat had led to cancer as well as the expression levels of the genes inquestion. The cells can also be cultured, grown and tested withdifferent combinations of chemotherapeutic agents for drug discovery andpersonalized medicine.

Detecting CTCs however is a challenging task because of their scarcityin blood samples, as few as single cell in multiple milliliter (mL)blood samples. The current favored approach to detecting whole cells inclinical and laboratory settings is flow cytometry, wherein labeledcells are detected as they flow in single file through an opticaldetector. This technology is used widely from vaccine analysis tomonitoring of AIDS. However, the high cost and large size of flowcytometers usually limits this testing approach to central facilitiesshared by many users. Furthermore, since the cells have to pass througha sensing portion of the flow cytometers in a single file manner,volumetric sample throughput is relatively low and cytometers need torun for long times to analyze large samples. For so-called “rare”cells—i.e., cells that are scarce in a fluid sample, such as CTCcells—relatively large volume samples may be required to find the cells.In this instance, the current flow cytometers can be prohibitivelyexpensive for frequent diagnostic usage.

Microfluidic cell detectors have been developed to overcome the cost andsize limitations of traditional flow cytometers in certain applications.These sophisticated systems can successfully interrogate small samples,on the order of μLs (microliters), but such systems have been found tohave limited capability for analyzing large samples, on the order ofmultiple mLs. Most microfluidic systems offer good performance inanalyzing small, microliter- or nanoliter-sized sample volumes. However,because of their micrometer dimensions, microfluidic “lab-on-a-chip”detectors need many hours to process large, milliliter-sized samplevolumes. Slow flow rates in microfluidic assays are usually aconsequence of the microscale dimensions of the sensing channels. Thesedimensions are necessary to increase the probability of a rare cell(i.e., a CTC) binding on the walls of the microchannels and in somecases to increase the signal-to-noise ratio of the underlying detectionmechanism. Thus, the prior microfluidic cell detectors can be generallyinefficient and can require prohibitively long analysis times foranalyzing the large volume samples necessary for the detection of raretargets like CTCs.

In one microfluidic detection system developed by the Toner and Habergroups of Massachusetts General Hospital, a lab-on-a-chip is populatedwith antibody-functionalized 100 μm diameter posts spaced 50 μm apart tocreate fluid flow paths. In another chip design, the posts were replacedwith a herringbone structure to actively assist mixing of the cells andincrease their probability to bind the functionalized walls. In thesestudies, flow rates used with clinical samples were on the order of 1 mLper hour, at which rate processing a typical 7.5 mL blood sample couldtake many hours. In order to reduce the fluidic transport times to amanageable level of minutes rather than hours, the flow rate throughthese prior systems would have to be increased by one or two orders ofmagnitude. In general the necessary modifications to prior microfluidicsystems can be problematic because: 1) the fluidic resistance of themicron-sized flow channels and the associated macro-to-micro connectionswould be very high; 2) a high flow rate through a small cross-sectionalarea would result in a high “linear speed” which would create shearstresses beyond levels that could be sustained by the antibody/cellbinding on the device wall and lead to detachment of the cells; and 3)too high a linear speed would detrimentally affect the captureefficiency of the target cells in the first place. Increasing the sizeof the channels would allow higher flow rates but this wouldsignificantly reduce the probability of the target cells' interactionwith the functionalized walls.

In the case of other microfluidic devices that use electronic detectiontechniques, larger dimensions would reduce the device's detectionsensitivity since most microdevices need some form of focusing oftargets onto a small sensor area for detection. Other researchers haveparallelized their microfluidic detectors (many micro-channels side byside) to overcome the throughput problem. However, the flow rates thatare used can be on the order of only 10 microliters (μLs) per minute,which can lead to hours of time to process the large volume samplesnecessary for CTC detection.

A high-throughput yet relatively simple and robust rare cell detectionsystem would be highly beneficial in many research and clinicalsettings. Therefore, a sensor apparatus is needed that can detect rarecells, such as CTCs, in whole blood in a high-throughput manner by whichsample fluids at rates of milliliters per minute (as opposed tomicroliters per minute) are processed to capture the contained cells.Such a system would also be highly useful in detecting various othertypes of cells, bacteria and spores present in sample fluids or in theenvironment.

SUMMARY

According to one aspect of the current teachings a fluidic systemincluding a detector component with micro-apertures is disclosed whichis configured to detect target entities bound by recognition elements,such as magnetic beads, in a high-throughput analysis for flow rates ofmilliliters per minute. In particular, a microfluidic detection systemis provided for detection of target entities, such as cells ormolecules, in a fluid containing a quantity of magnetic beads and aquantity of target entities bound to one or more magnetic beads, inwhich each target entity bound to a magnetic bead has a smallesteffective dimension greater than a smallest effective dimension of eachmagnetic bead. In one aspect, the system comprises a detector componentincluding a body defining a reservoir and a sensor chip in the form ofan apertured plate disposed within the reservoir and separating thereservoir into a first chamber and a second chamber. The plate includesa plurality of micro-openings, each opening having smallest effectivedimension greater than the smallest effective dimension of each magneticbead and less than the smallest effective dimension of each targetentity bound to a magnetic bead. In one aspect, the first chamber of thereservoir has a first inlet and a first outlet, in which the first inletis fluidly connectable to a source of the fluid containing the targetentities, while the first outlet is fluidly connectable to a collectionvessel.

The detector component further includes a magnet disposed relative tothe reservoir so that the second chamber of the reservoir and theapertured plate are situated between the magnet and the first chamber ofthe reservoir. The magnet configured to generate a magnetic forcesufficient to attract magnetic beads in the first chamber of thereservoir to the second chamber of the reservoir. The microfluidicdetection system further comprises a pump for continuously flowing thefluid from the source of the fluid containing the target entitiesthrough the first chamber of the reservoir.

Another detector component includes a second magnet situated above thefirst chamber of the reservoir. The second magnet may be positionedoutside the detector component so that it can be moved relative to thereservoir and removed. The polarity of the second magnet can be arrangedto complement the polarity of the magnet below the apertured plate andthe second chamber so that the two magnets cooperate to direct entitiesbound to magnetic beads into the second chamber. The second magnet maybe positioned across the face of the detector component so that themagnetic field can move side to side in the chamber, thereby dislodgingmagnetic beads that might be trapped on the apertured plate. The secondmagnet may be controllable to engineer the magnet field applied to themagnetic beads and entities bound to the beads.

A method is provided for detecting target entities in a fluid containinga quantity of magnetic beads and a quantity of target entities bound toone or more magnetic beads, in which each target entity bound to amagnetic bead has a smallest effective dimension greater than largesteffective dimension of each magnetic bead. In one aspect, the methodcomprises: continuously flowing the fluid through a first chamber of areservoir separated from a second chamber of the reservoir by anapertured plate, each opening in the plate having a smallest effectivedimension greater than the smallest effective dimension of each magneticbead and less than the smallest effective dimension of each targetentity bound to a magnetic bead; and applying a magnetic force beneaththe apertured plate sufficient to draw magnetic beads not bound to atarget entity through the apertures into the second chamber andsufficient to hold the target entities bound to one or more magneticbeads against the surface of the apertured plate within the firstchamber of the reservoir.

The target entities may be specific cells or molecules, such as smallmolecules, or other biological entities capable of binding to thefunctionalized beads. Thus, in some methods only certain cells orcertain molecules are detected, with appropriate functionalization ofthe magnetic beads. In another method, differently functionalized beadsmay be introduced into a fluid sample to bind with both cells andmolecules in the sample. In this method bound cells can bedifferentiated from bound molecules within the detector component sothat the captured cells and molecules can be separately analyzed andprocessed, all from the same sample. Different metrics can be appliedbased to the combined captured entities, such as the ratio of capturedcells to captured molecules.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic view of a microfluidic detection system accordingto the present disclosure.

FIG. 1B is a schematic view of a portion of the system shown in FIG. 1Amodified in accordance with an alternative embodiment.

FIG. 2 is a side cross-sectional view of a detector component accordingto the present disclosure for use in the detection system shown in FIG.1.

FIG. 3 is a plan view of a sensor chip micro-perforated plate for use inthe system shown in FIG. 1A.

FIG. 4 is a side representation of the operation of the sensor chipshown in FIG. 3 to capture target entities and extract unbound magneticrecognition elements.

FIGS. 5A-D are schematic views of the microfluidic detection system ofFIG. 1A, showing fluid flow paths in different stages of operation ofthe system.

FIG. 6 is a bright-field microscopic picture of target entities capturedon a sensor chip in accordance with a disclosed embodiment.

FIG. 7 is a side cross-sectional view of another detector componentaccording to the present disclosure.

FIG. 8 is a side representation of a detection process using thedetector component of FIG. 7.

FIG. 9 is a perspective representation of a detection process applied tothe detection surface of the detector component of FIG. 7.

FIG. 10 is a side cross-sectional view of a further detector componentaccording to the present disclosure.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and described in the following written specification. It isunderstood that no limitation to the scope of the invention is therebyintended. It is further understood that the present invention includesany alterations and modifications to the illustrated embodiments andincludes further applications of the principles of the invention aswould normally occur to one of ordinary skill in the art to which thisinvention pertains.

The present disclosure provides a micro-fluidic system with a detectorcomponent having an apertured plate or chip configured to providehigh-throughput analysis of mLs (milliliters) per minute of a fluidsample flowing through relatively large (millimeter (mm) as opposed tomicrometer (μm)) flow channels. The fluid sample may be a bodily fluid,including but not limited to whole blood, processed blood, serum,plasma, saliva and urine, or environmental fluids, including but notlimited to fluid samples from rivers, sewage lines, water processingfacilities and factories. Using functionalized beads capable of bindingto target entities in three dimensions, the system eliminates the needfor chemical affinity-based binding of the cells to a stationarytwo-dimensional chip surface. The system uses both convective fluid flowto assist in mass transport of target cells bound by functionalizedmagnetic beads, and magnetic sieving of the bound cells onto a plate orchip with micro-apertures which captures the cells but allows freemagnetic beads (i.e., those not bound to target entities) to passthrough the apertures. In certain embodiments the magnetic beadssimultaneously serve to: 1) affinity-based bind specific target cells;2) magnetically transport the bound cells to the plate or chip surface;and 3) generate recognition or labeling signals for detection. The novelfluidic system of the present disclosure can analyze large amounts ofsample fluids, including clinically significant amounts of bodily fluidssuch as whole blood, in a relatively short amount of time.

FIG. 1A depicts a schematic of a micro-fluidic system 10 according toone embodiment of the present disclosure. The system 10 includes adetector component 12 having a first flow segment 13 and a parallelsecond flow segment 14. The first flow segment 13 is provided with aninlet 13 a and an outlet 13 b, in which the inlet is connected by aninlet conduit 35 to a sample fluid source S. The outlet 13 b isconnected by outlet conduit 36 to a collection vessel CC, which issuitable for collecting and storing target entities isolated by themicrofluidic detection system 10. The second inlet 14 a is connected byan inlet conduit 38 to a source of a buffered or physiologically inertor non-reactive solution B. The second outlet 14 b is connected by anoutlet conduit 39 to a collection vessel BC for collecting bufferedsolution exiting the detector component 12.

In one embodiment a pump 40 is provided for flowing fluid from thesample source S through the first flow segment 13 of the detectorcomponent 12. In the embodiment shown in FIG. 1 the pump 40 isintegrated into the outlet conduit 36 to draw fluid through the detectorcomponent. However, it is contemplated that the pump 40 may be situatedwithin the inlet conduit 35 as desired. The embodiment of FIG. 1Afurther includes a second pump 41 that is integrated into the outletconduit 14 b to flow the buffer fluid from source B through the secondflow segment 14 of the detector component 12. The pump 41 may beprovided in the inlet conduit 38 to draw buffer fluid from the source Band pump it through the second flow segment 14. Alternatively the samepump, such as pump 40, may be used to pump both the sample fluid fromthe source S through the first flow segment 13 and the fluid from sourceB through the second flow segment 14, as depicted in the enlargedsegment shown in FIG. 1B. In this embodiment the two outlet conduits 36,39 are connected to the pump 40 through a bi-directional valve V6 thatis controllable to selectably connect one or the other outlet for flowthrough the pump, according to a fluid flow protocol discussed herein.

Returning to FIG. 1A, the microfluidic system 10 incorporates bypasslines that are selectively activated according to a fluid flow protocoldescribed below. A bypass line 42 is provided between the buffer sourceB and the inlet conduit 35 to the first flow segment 13 of the detectorcomponent. A valve V1 is operable to control flow of buffer solutionfrom the source B into both inlet conduits 35, 38, while a valve V2 isoperable to control flow of buffer solution into the first inlet conduit35.

In a second bypass path, a bypass line 43 is connected between the firstoutlet conduit 36 and the first inlet conduit 35. This bypass line thusreturns fluid exiting the first flow segment 13 of the detectorcomponent back to the inlet 13 a for the first flow segment. A valve V3controls fluid flow through the second bypass line 43. A third bypasspath includes the bypass line 44 from the second outlet conduit 39 tothe source S containing the sample fluid. A valve V4 is configured todirect the flow of buffer fluid exiting the second flow segment 14either to the buffer collection vessel BC or to the third bypass line44. The system 10 is provided with a control module 45 that is operableto control the pump(s) 40 (and 41 if present) as well as the valves tocontrol the flow of fluids through each flow segment 13, 14 of thedetector component 12, according to a flow protocol described herein.

The microfluidic system 10 may be configured to accept sample fluidsfrom multiple sources S_(i). The multiple sources S_(i) may be connectedin series or in parallel, with appropriate valving to connect theparticular source to the inlet conduit 35 to the first flow segment 13of the detector component. The system may be further modified to includeadditional detector components 12 i connected to the outlet conduit 36from the first flow segment 13, by way of a control valve V5.

The flow of sample fluid and buffer solution through the system 10, andparticularly through the detector component 12, has thus far beendescribed. Details of the detector component 12 and its function areillustrated in FIGS. 2-4. Turning first to the cross-sectional view inFIG. 2, the detector component 12 includes a body 15 that may be in theform of two halves 15 a, 15 b that are combined to form the completecomponent. The body 15 defines a reservoir 16 that is separated into afirst chamber 16 a and a second chamber 16 b by a sensor chip in theform of a micro-apertured plate 18. The first chamber 16 a is incommunication with the first flow inlet 13 a and the first flow outlet13 b and thus forms the first flow segment 13 of the detector component.A first inlet channel 22 communicates between the first chamber 16 a andthe inlet 13 a, while a first outlet channel 23 communicates with theoutlet 13 b. In the illustrated embodiment, the two channels are angledtoward the reservoir 16, or defined at a non-planar angle relative tothe reservoir, so that the target entities and sample fluid do notaccumulate within the channel 22 or inlet conduit 35.

The second chamber 16 b is in communication with the respective secondinlet and outlet 14 a, 14 b to form the second flow segment 14 of thedetector component. An inlet channel 25 communicates between the secondchamber and the second inlet 14 a, which an outlet channel 26communicates with the second outlet 14 b. The two channels 25, 26 can beangled but need not be since the second chamber 16 b is connected to thebuffer solution source B and no target entities flow through this secondfluid flow path 14.

The reservoir 16 may be open at one side of the detector component 12,with the reservoir opening sealed and closed by a window or viewingpanel 20. The viewing panel 20 is oriented to provide an unobstructedview of the apertured plate 18 within the reservoir 16. In oneembodiment the viewing panel 20 is optically transparent to permitdirect visualization of the surface of the apertured plate.

The sensor chip or apertured plate 18 may be supported by a platemounting 28, formed around the perimeter of the plate that is trappedbetween the two body halves 15 a, 15 b when they are coupled together. Aseal 29 may be provided on one or both sides of the plate mounting 28 toensure a fluid-tight seal between the two chambers 16 a, 16 b. Detailsof the apertured plate are shown in FIGS. 3-4. In particular, the plate18 includes an upper surface 51 and a plurality of micro-sized aperturesor openings 50 defined therethrough. In one embodiment the openings aregenerally uniformly sized and have a largest effective dimension d thatis less than the smallest effective dimension of a target entity T (FIG.4) that is to be detected. Moreover, the smallest effective dimension dof the plate openings is greater than a largest effective dimension ofmagnetic recognition elements M. For the purposes of the presentdisclosure, the term “effective dimension” refers to a dimension of aparticular element measured along a particular axis. For a circularopening in the plate or a spherical magnetic bead, the smallest andlargest effective dimensions are the same and are simply the diameter ofthe opening or bead. For an oblong opening, the largest effectivedimension is the length of the opening along its long axis, while thesmallest effective dimension is the width along the short axis. Thetarget entities that are cells, the cells may not exhibit a uniformthree-dimensional shape, (such as a sphere) so the cell will have adifferent dimension depending upon the axis of measurement. For cells ofthis type, the term “smallest effective dimension” refers to thesmallest of those measurements. Thus, the relative effective dimensionsof the plate openings are such that a magnetic bead can always passthrough any opening no matter how the bead is oriented, while a targetentity can never pass through any opening regardless of how it isoriented.

The microfluidic system 10 is configured to detect and isolate targetentities T that are bound to recognition elements M. Thus, the fluidsource S holds a sample fluid that contains target entities, forinstance a blood sample of a patient that contains circulating tumorcells (CTCs) or a sample that has exemplary tumor cell lines such aslymph node carcinoma of the prostate cells (LNCaP) or ovarian cancercells (IGROV). The sample may alternatively or in addition includemolecular target entities, such as the PSA molecule used to detectprostate cancer. The fluid sample further contains recognition elementsin the form of magnetic beads M that bind to the target entities.Details of the target entities and recognition elements will follow, butwith respect to the openings 50 in plate 18 it can be appreciated thatthe size of the openings is calibrated so that any free magnetic beads M(i.e., beads that have not bound to a target entity T) will pass freelythrough the opening, such as the beads M_(X) on the right side of theplate in FIG. 4. On the other hand, where the target entities are cells,the openings 50 are sized so that the target cells T cannot passtherethrough, with or without any magnetic beads bound thereto, such asbeads M_(B).

The significance of the magnetic beads M can be appreciated by referringback to FIG. 2. In particular, the body 15 of the detector component 12includes a magnet 32 mounted within a cavity 31 beneath the secondchamber 16 b. In particular, the magnet 32 is positioned so that themagnetic force attracts magnetic beads M within the first chamber 16 atoward the apertured plate 18. It is this magnetic force that pulls thefree magnetic beads M_(X) through the openings 50, as illustrated inFIG. 4, even while the beads are under the influence of a fluid flow Fthat is substantially parallel to the surface 51 of the plate 18. Thissame magnetic force also attracts beads M_(B) that are bound to a targetentity T, which are also under the influence of the parallel fluid flowF. However, since the target entity T is too large to pass through anyopening the magnetic force serves to hold the bound target entityagainst the surface 51 of the apertured plate 18. In certain instancesthe target entity T is large enough relative to the magnetic beads M tohave several beads M_(B) bound to the cell. As depicted in FIG. 4, someof the bound beads M_(B) extend partially into an opening 50. The beadsM_(B) are held within the opening by the magnetic force, which not onlyholds the bound target entity T to the plate surface 51 but alsorestrains or “locks” the cell against translating along the surface orbeing washed away under the influence of the fluid flow F. Thus, thebound beads M_(B) not only capture target entities but also help preventthe captured target entities from bunching up or collecting at theoutlet end of the first chamber 16 a.

The magnet 32 is calibrated relative to the magnetic beads M to exert amagnetic force sufficient to pull the beads toward the apertured platebut not so strong as to break the bound beads M_(B) away from a targetentity T captured on the plate surface 51. The magnetic force is alsosufficiently strong to pull the beads and target entities out of thefluid flow F that tends to propel the beads and cells in a flow pathparallel to the surface 51 of the sensor chip plate 18. In a specificexample the magnet is a NdFeB Cube Magnet (about 5×5×5 mm) with ameasured flux density and gradient of 0.4 T and 100 T/m, respectively.Other magnets are envisioned including but not limited to larger orsmaller permanent magnets made of various materials, and electromagnetsthat are commercially available or manufactured using standard ormicrofabrication procedures and that are capable of generatingtime-varying magnetic fields. In the illustrated embodiment of FIG. 2the magnet 32 is housed within a cavity 31 formed in the bottom half 15b of the housing. However, the magnet may be affixed to or supportedrelative to the outside of the detector component 12 provided that it isoriented in a manner to draw the magnetic beads M from the first chamber16 a to the second chamber 16 b. It is further contemplated that themagnet 32 may be associated with the detector body 15 so that thedistance of the magnet from the apertured plate 18 may be varied tothereby vary the magnetic force applied to the magnetic beads in thefirst chamber 16 a. The magnetic force may thus be calibrated to aparticular magnetic bead. In addition, the magnet 32 may be moved toremove the magnetic force entirely according to a flow protocol for themicrofluidic system 10. Removal of the magnetic field can facilitate theremoval of captured target entities from the plate surface so that thetarget entities may be transported or flushed to a separate collectionvessel CC.

In another embodiment, a magnetic field may be applied from the top ofthe detector component 12 or directly above the surface 51 of the sensorchip plate 18. This magnetic field may thus “levitate” the capturedtarget cells off the surface 51 to further facilitate their removal. Itis contemplated in this embodiment that the magnetic field of the magnet32 is disrupted as described above so that the magnetic field appliedfrom the top of the component does not “compete” with the originalcapturing magnetic field.

In a further embodiment, a detector component 70 includes a magnet 82similar to the magnet 32 described above that is positioned below theapertured plate 85 and a second magnet 84 provided above the detectorcomponent 70 and the viewing panel 80. The polarity of the second magnetmay complement the polarity of the first magnet 82, as depicted in FIG.7, so that the magnet force applied to the magnetic beads is alwaystoward the second chamber 79 defined in the body 75. In one aspect, thesecond magnet 84 may be movable relative to the detector component atleast so that the magnet can be removed during post-processing of thecaptured target entities. The second magnet 84 may further be movable sothat the magnet can be moved in a sweep pattern or rotated relative tothe chamber 76 and apertured plate 85 to facilitate movement of un-boundmagnetic beads, for instance. For example, in some cases magnetic beadsmay settle on the apertured plate 85 rather than flow through theapertures into the second chamber 79, such as might occur if the fluidsample flow through conduits 77 and 78 is not sufficiently high toprevent the beads from settling on the plate. The beads may be agitatedby movement of the second magnet 84 so that they will eventually find anaperture and fall into the second chamber. Thus, the second magnet maybe moved back and forth over the plate or in a rotating or circularpattern over the detector component 70. Movement of the magnet 84 may beaccomplished by a motor and may be provided with a controller thatallows selection of a particular sweep pattern for the magnet. Themagnets 82 and 84 may be electromagnets that can be activated ordeactivated as desired. Moreover the electromagnets may be configured toreverse polarities as part of a technique for controlling movement ofthe magnetic beads and ligand-bound entities.

In another aspect, the second magnet 84 may be controllable ormanipulable to engineer the magnetic field and magnetic forces appliedto the bound and unbound magnetic beads. For instance, the magneticfield may be controlled to direct magnetic beads onto differentpositions on the detector surface 88 of the lower portion 86 of thedetector body. The polarity of both magnets 82, 84 may also be modifiedfrom that depicted in FIG. 7 including conditions in which likepolarities face each other.

As explained above, the pump(s) 40 (41) and the valves V1-V5 arecontrolled according to a flow protocol adapted to; a) prepare thedetector component 12 to receive a fluid containing bound targetentities; b) capture the bound target entities; c) flush unboundmagnetic beads; and d) extract captured target entities. In a first stepof the protocol, the system is primed with an non-reactive or bufferedsolution from source B. The solution from source B is preferablynon-reactive to the target entities T, to the recognition elements ormagnetic beads B, and to any ligands, antibodies, aptamers, peptides,low molecular weight ligands, or antigens used to functionalize and bindthe recognition elements. In a specific embodiment the solution may be aphosphate buffered saline (PBS). Referring to FIGS. 1A and 5A, thereservoir 16 is initially flooded with PBS by opening valve V1, movingvalve V4 to close the bypass line 44 but open the flow path to thecollection container BC, and moving valve V2 to close the inlet conduit35 to the sample source S and open the conduit to the bypass line 42.Valves V3 and V5 are closed so that all of the fluid exiting thedetector component 12 is fed to the buffer collection container BC. Thebuffered solution PBS flows freely through both chambers 16 a, 16 b andthrough the apertured plate 18 so that all fluid flows through thesecond outlet channel 26 and second outlet 14 b into outlet conduit 39and collection container BC. In certain embodiments it may be desirableto open valve V5 to pump PBS from chamber 16 a into collection vessel CCin order to avoid any pressure increase within the chamber. The pump 41is thus activated to control the flow of PBS through both flow segments13, 14. In the alternative configuration of FIG. 1B, the pump 40provides the motive force for fluid flow with the valve V6 open to bothoutlets 13 b, 14 b but with pump discharge to only the second outletconduit 39. It can be appreciated that this initial flow of PBS throughthe system will purge the air from the reservoir and channels.

With the detector component primed, the buffered solution fluid circuitis deactivated by closing the valves V1 and V4, closing the bypass line42 at valve V2, and deactivating pump 41. The first chamber 16 a is nowready to receive the sample fluid from source S by opening valve V2 tothe inlet conduit 35 and valve V5 to the collection vessel CC, asillustrated in FIG. 5B. Pump 40 is activated to draw the sample fluidfrom the source S through the detector component 12, and moreparticularly to pull the sample fluid through the first inlet 13 a intothe first chamber 16 a. The magnet 32 is activated to pull the magneticbeads M to the apertured plate 18, as depicted in FIG. 4. It can beappreciated that the rate of flow F of the sample fluid is calibrated sothat the fluid pressure does not overcome the magnetic force. By way ofnon-limiting example, the pump 40 may be configured to produce a flowrate of several mLs/min, which is significantly faster than the mLs/hourrates of prior microfluidic systems. In one specific embodiment theinlet and outlet channels 22, 23 may have a smallest effective dimensionof 0.5 mm so that a 1 mL/min flow rate may generate a linear flowvelocity of about 3 mm/sec in the channels and about 0.7 mm/sec throughthe reservoir 16. These linear velocities are nearly 100 fold lower thanvelocities believed to cause damage to target entities T. However, atthis flow rate a typical 7.5 ml fluid sample may pass through thedetector component 12 in 7.5 minutes or less. Similarly, a 3 mL/min flowrate would indicate a passage of a 7.5 mL sample in about 2.5 minutes.

The target entities and magnetic beads are under the influence of fluidflow that attempts to wash them away from the sensor chip surface aswell as a magnetic force that attempts to draw them to the chip surface.The magnetic force produced by the magnet 32 can thus be calibrated tocounteract the influence of the fluid flow F. In other words, a greaterflow rate may be accomplished by increasing the magnetic force, since agreater force is required to dislodge the cells and beads from the fluidflow. A limiting factor to the strength of the magnetic field generatedby the magnet 32 is that the magnetic force cannot be great enough todisassociate the magnetic beads B from the bound target entities T orgreat enough to damage the target entity as the beads are pulled by themagnetic force.

As the fluid sample flows through the first chamber 16 a the magnetattracts the recognition elements M to the plate 18 and lower secondreservoir 16 b. As explained above, most of the unbound beads M_(B) willpass through the openings 51 and into the lower reservoir 16 b wherethey are held in place by the magnetic force. Likewise, the bound targetentities T will be captured against the surface 51 of the aperturedplate 18 so long as the magnetic force is present. The remaining samplefluid, less the captured target entities, may be delivered to thecollection vessel CC. Alternatively, the valve V5 may be closed and thevalve V3 opened to allow the sample fluid discharged from outlet 13 b tobe returned to the inlet 13 a via bypass line 43, as reflected in thediagram of FIG. 5C. The use of the bypass can account for any boundtarget entities or any unbound magnetic beads that escape capture withinthe reservoir 16. The sample fluid may be continuously recirculated fora period of time deemed sufficient to capture all of the bound targetentities.

It can be appreciated that at the end of the this second stage of theflow protocol all or at least a majority of the bound target entities Tin the sample fluid have been captured against the surface 51 of theapertured plate 18 within the upper first chamber 16 a. Likewise, all orat least a majority of the unbound magnetic beads R_(B) have been pulledthrough the openings and are collected in the lower second chamber 16 b.The captured target entities are thus available for viewing through theviewing panel 20 in order to count the number of target entities, forinstance. It is contemplated that in a typical procedure the targetentities will be rare or at an extremely low concentration within asample (e.g., CTCs in a blood sample). Thus, the number of capturedcells may be very low but easily discernible on the apertured plate. Inone approach the captured cells may be viewed by bright-fieldmicroscopy. In addition, the captured cells may be further labeled withfluorescent reporters and visualized using fluorescent microscopy.Alternatively or in addition, the magnetic beads may be functionalizedwith a visual indicator, such as with fluorescent labeling. The magneticbeads may be visualized using fluorescence microscopy. Since the targetentities are typically bound to a number of magnetic beads thefluorescent image of the beads will reveal the presence of the boundtarget entities. An example of captured target cells is shown in thebright-field microscopy image in FIG. 6. In this image the capturedcells are clearly visible. The cells are MCF-7 (breast cancer cells)that are bound to magnetic beads functionalized with anti-EpCAMantibodies in a known manner. It can be noted that while the greatmajority of the several million unbound magnetic beads in the samplepassed through the plate openings, some unbound magnetic beads are alsopresent on the plate surface. However, it is apparent that the presenceof these few beads does not interfere with a clear view of the capturedtarget cells. In the specific example shown in FIG. 6 the openings havea diameter (or smallest effective dimension) of 5 μm and the magneticbeads have a diameter of 300 nm.

In order to improve visualization of the collected cells, the reservoirmay be washed to eliminate the sample fluid and other cells that mightvisually interfere. In this instance, the magnetic field is maintainedwhile the buffered solution from source B is flowed through the upperand lower portions of the reservoir. The washing cycle may be conductedin the same manner as the initially preparation cycle described above,namely by opening the two chambers 16 a, 16 b to the PBS solution,closing the valve V5 and opening the valve V4 to the buffer collectionvessel BC. Since the magnet remains in position during this washingcycle the target entities will remain captured on the apertured platewithin upper chamber 16 a and the unbound magnetic beads will remaincollected within the lower chamber 16 b.

The microfluidic system 10 disclosed herein is also capable ofcollecting the captured target entities T as well as recovering themagnetic beads M. In one approach, the buffered solution (PBS) is flowedonly through the upper first chamber 16 a with the magnetic fieldremoved. Thus, as shown in the diagram of FIG. 5D, the valve V1 iscontrolled to close flow to the inlet 14 a but open to conduit 35 andvalve V1. Valve V1 is controlled to prevent flow from the source S butaccept the PBS flow from source B. Valves V3 and V4 are closed but valveV5 is open to the collection vessel CC. In the absence of the magneticfield the bound target entities are easily dislodged from the aperturedplate. The flow of PBS can wash the target entities through the outletconduit 23 and into a collection vessel CC. Since the second inlet 14 aand outlet 14 b are closed there is no fluid flow through the lowersecond chamber 16 b. Thus, the collected beads M remain pooled at thebottom of the reservoir 16 even as the target entities are washed away.Alternatively, the magnetic field may be adjusted to reduce the magneticforce experienced by the bound target entities to a level sufficient tobe overcome by fluid pressure from the PBS flowing through the upperchamber 16 a. Since the pooled unbound beads in the lower chamber arecloser to the magnet, the magnetic force is sufficient to hold the beadsin place. Once the target entities have been removed and collected themagnetic field can be removed and valves V1 and V4 opened to flow PBSthrough the lower chamber to wash the unbound beads into the collectionvessel BC. Alternatively, the valve V4 can be activated to open thebypass line 44 to redirect the unbound beads back to the sample sourceS. In this instance the unbound beads may be incubated to bind with anypreviously unbound target entities in the original source S oradditional sources Si.

As previously described, the microfluidic system 10 may includeadditional detector components 12 i that may be brought on line by viathe valve V5. In certain protocols is may be contemplated that thesample fluid will flow continuously from the first detector component 12to each successive detector component 12 i before flowing to thecollection vessel CC.

It is contemplated that the conduits and valves be formed of chemicallyinert materials. In a specific example the conduits 35, 36, 38, and 39,and the bypass lines 42-44 may be tubing such as 1/16 inch Cole-Parmertype tubes or other chemically-inert tubing. In certain procedures thesource of target entities may be a conventional 7.5 mL whole bloodsample in which the targeted cells have already been bound torecognition elements, such as magnetic beads. For a typical flowprotocol, the buffer source B may be a 10 mL PBS reservoir or larger. Incertain procedures the sample may be a processed blood sample in whichthe red blood cells have already been removed by means of lysing or bymeans of commercially available tubes, such as BD Vacutainer CellPreparation Tubes. In other procedures the sample may be other bodilyfluids such as urine, or may be water or other fluid samples collectedfrom environmental or industrial sources.

In one embodiment the top and the bottom halves 15 a, 15 b of thedetector component body 15 are machined out of acrylic and fastened byscrews in a manner that sandwiches the apertured plate 18 and seal ring29 to form a fluid tight seal between the chambers. However, othermanufacturing and material are also envisioned, including but notlimited to molded plastic formed in a plastic molding operation.

In one embodiment, the sensor chip apertured plate 18 is about a 15 mmby 15 mm silicon-on-insulator (SOI) wafer having a thickness of about0.5 mm. The openings 50 may be limited to a predetermined active area ofthe detector component of about 10 mm by 10 mm. The array of openings(such as checker-board arrangement) may be defined on the wafer usinglithography and then the holes formed by reactive ion etching of thefront side of the wafer. Individual sensor chips may be defined byreactive ion etching of the back side of the wafer followed by HFetching of the insulating oxide. Alternatively, grooves may be definedusing lithography and etched into the front side of a silicon wafer byreactive ion etching followed by coating with a thin layer of nitride.The nitride on the backside of the wafer can be patterned usinglithography etched to define individual chips. Finally, the siliconwafer can be etched in the opening array pattern using reactive ionetching or potassium hydroxide, and the remaining nitride layer can beremoved by etching. As discussed above, the openings have a smallesteffective dimension that is sufficiently small to trap target cell-boundmagnetic beads, yet sufficiently large to allow free magnetic beads thatare not bound to the target entities to pass therethrough. In a specificexample, the target entities are cells, and particularly CTCs, so theopenings need to be smaller than the targeted CTCs but larger than thebeads. For example, the average size of a lymph node carcinoma of theprostate cells (LNCaP) or ovarian cancer cells (IGROV) is about 20 μmwhile the size of a certain type of magnetic bead may be about 1 μm.Hence, 3 μm openings will be large enough to easily pass a free bead buttoo small to let a CTC through. In a specific embodiment, the openingsmay be provided at about 30% packing density, which can result in about14 openings underneath a 20 μm cell. Furthermore, if each cell is boundby multiple magnetic beads (as depicted in FIG. 4), each bead is pulledby the magnetic force so that the target cell is pulled down in multiplelocations, making it even more difficult for a cell to pass through asingle opening. The openings are also configured to trap the cell-boundmagnetic beads to “lock” the target cells from moving horizontally,preventing them from being washed away from the surface of the plate bythe fluid flow. In the embodiment illustrated in FIGS. 3-4, the openings50 are shown as having a circular or cylindrical with a diameter.However, the openings may have other shapes, such as a conical bore oran oblong opening in the direction of the fluid flow F provided that thesmallest effective dimension of the opening meets the dimensionalrequirements set forth above. In an alternative embodiment, the plate 18may be configured with micro-grooves each having a width equal to thesmallest effective dimension d discussed above sized so that the targetcells cannot enter the micro-grooves but the much smaller magnetic beadscan. The openings 50 may have other shapes to facilitate manufacturing.For instance, the openings may have a polygonal rather than circularshape, with the dimension between sides of the polygon defining thesmallest effective diameter of the openings. Certain fabricationtechniques, such as lithographic mask techniques, can form polygonalholes more easily than circular holes. The openings may also beconfigured to modify the fluid flow in the vicinity of the aperturedplate. For instance, an opening that is sloped across the thickness ofthe plate from the inlet 77 to the outlet 89 may modify the fluid flowto draw the bound targets into the openings. The downstream end of theopening (i.e., nearer the outlet) may be raised to help hold the targetswithin the openings until they are propelled through the opening by thecontinued fluid flow. The goal in this configuration of the aperturedplate and openings is to facilitate transfer of bound targets throughthe apertured plate and into the lower chamber 79.

The apertured plate 18 may be coated or passivated with aphysiologically inert material, such as bovine serum albumin (BSA) orpoly ethylene glycol (PEG). Since the system according to the presentdisclosure does not utilize chemical binding between a functionalizedtarget cell and the plate, the surface of the plate can be, and ispreferably, non-reactive.

In accordance with the present disclosure, the target cell-to-magneticbead binding is the only aimed affinity binding step, since the detectorcomponent 12 does not rely on chemically binding the target cells to aportion of the component. Magnetic beads are functionalized in manyconventional ways, including with appropriate monoclonal or polyclonalantibodies (including but not limited to EpCAM antibodies), aptamers orshort peptides that can bind to specific target cells. In an alternativefunctionalization strategy, low molecular weight ligands (e.g. 2-[3-(1,3-dicarboxy propyl)-ureido] pentanedioic acid or “DUPA” for prostatecancer cells, and folic acid for ovarian cancer cells or other cancercells that over-express the folate receptor on their surfaces includinglung, colon, renal and breast cancers) are used to promote binding tocertain cells, most particularly CTCs. Specifically, low molecularweight ligands (e.g. DUPA and folate) can be produced with a functionalgroup (amino, n-hydroxy succinamide (NETS), or biotin depending on thefunctional group on the magnetic bead to be used) with a PEG chain inbetween the low molecular weight ligand and the functional group tosuppress nonspecific binding to the beads.

Functionalized beads are available from a variety of vendors withchemically reactive groups. Magnetic beads are also available in a widerange of sizes (from 100 nm to 5 μm, for example) that can be selectedbased on the dimensions of the target cell to which the beads are bound.In one embodiment NETS-coated 1 μm beads can be the starting point fromChemagen. For these beads, the PEG chain will be terminated with anamino group for covalent linkage to the NETS group on the bead. Thebeads can also be tested from other vendors with other functionalgroups, and can be terminated with the PEG chain and functional groupaccordingly. The beads can also be functionalized with fluorescentmolecules using the appropriate chemistry for the functional group. Forthe example of a low molecular weight ligand, DUPA-PEG-amine andfolate-PEG-amine molecules can be synthesized which can be reacted withthe beads before fluorescent labeling. Thereafter, the desired amount ofreactive fluorescent dye (fluorescein, for example) can be reacted withthe beads, after which residual NETS groups (or other activated moietieson the beads) can be passivated by reaction with glucosamine (or anotherappropriate molecule for neutralizing the activated moiety on thebeads). The ratio of folate-PEG-amine or DUPA-PEG-amine to fluorescentdye and passivating molecule can be optimized, as needed. Similarly,antibodies (such as the epithelial cell adhesion molecule or “EpCAM”)can be immobilized on beads. One commercially available example, inwhich the functionalized magnetic beads are magnetic beadsfunctionalized by attachment to a monoclonal antibody against the humanEpithelial Cell Adhesion Molecule (EpCAM), such as Dynabeads® EpithelialEnrich, commercially available from Invitrogen. For example antibodiescan be covalently linked to NETS-coated beads, or can be linked tostreptavidin-coated beads via a biotin. Various other functionalizationschemes can also be used including but not limited to carboxyl groups,thiols, and silanes. Alternatively, the beads may only have therecognition elements to bind and trap the cells on the plate surface butlack the fluorescent reporters which could be introduced separately tobind directly to the captured cells. In one procedure, fluorescentlylabeled antibodies (e.g. cytokeratin), low molecular weight ligands,peptides or aptamers can be separately exposed to the captured cells.

The microfluidic system 10 and detector component 12 disclosed hereinare particularly suited to detection of cancer cells bound to magneticbeads. Many techniques are available for functionalizing and bindingmagnetic beads to target cells such as CTCs. Exemplary procedures aredisclosed in the published the following publications: T. Mitrelias, etal., “Biological cell detection using ferromagnetic microbeads,” Journalof Magnetism and Magnetic Materials, vol. 310, pp. 2862-2864, March2007; N. Eide, et al., “Immunomagnetic detection of micrometastaticcells in bone marrow in uveal melanoma patients,” Acta Ophthalmologica,vol. 87, pp. 830-836, December 2009; Yu et al., “Circulating tumorcells: approaches to isolation and characterization”, The Journal ofCell Biology, Vol. 192, No. 3, pp. 373-382 (Feb. 7, 2011); Hayes &Smerage, “Circulating Tumor Cells”, Progress in Molecular Biology andTranslational Science, Vol. 95, 2010, pp. 95-112; Alexiou et al.,“Medical Applications of Magnetic Nanoparticles”, Journal of Nanoscienceand Nanotechnology, Vol. 6, 2006, pp. 2762-2768; and Ito, et al.,“Medical application of functionalized magnetic nanoparticles”, Journalof Bioscience and Bioengineering, Vol. 100, 2005, pp. 1-11, thedisclosure of each publication being incorporated herein by reference.

Publications disclosing exemplary procedures for synthesis andmodification of folate and DUPA are identified in the Appendix. Any ofthe procedures and methods disclosed in these publications may besuitable for binding magnetic beads to select CTCs that may besubsequently captured by the detector component 12 disclosed herein.Functionalization of magnetic beads with folate is fully described inreferences listed in the Appendix, therefore one having ordinary skillin the art is enabled to functionalize similar magnetic beads with DUPAas further described in the listed references. The DUPA molecule itselfis also fully described in references listed in the Appendix andelsewhere in this specification.

The ability to attract bead-bound cancer cells to a solid surface (i.e.,without any openings) has been verified in experiments using MCF-7 cells(breast cancer cell line) attached to magnetic beads via EpCAMantibodies. The target cells were flowed with volumetric flow rateshigher than 2 mL/minute and were successfully magnetically attracted tothe solid surface during the flow. The target cells have a smallesteffective dimension of about 20 μm so in another experiment the targetcells were flowed over a plate having openings with an effectivediameter of 5 μm. Intact cancer cells were observed on the plate usingdual surface (cytokeratin) and nuclear (DAPI) staining. In theseexperiments 9 out of 10 target cells were detected in a 12 mL sample,for a 90% cell recovery.

The operation of the microfluidic detection system 10 disclosed hereincan be controlled through a master controller 45. The controller may bea microprocessor configured to follow a controlled flow protocolaccording to a particular target cell, recognition element and samplesize. The master controller may incorporate a reader to read indiciaassociated with a particular sample or samples, and automatically uploadand execute a predetermined flow protocol associated with the particularsample.

The controller 45 may also be configured to allow user-controlledoperation. For instance, the flow rate for a particular targetcell-magnetic bead combination can be optimized by increasing the flowrate of a bound target cell sample until it is no longer possible toattract beads to the surface 51 of the apertured plate 18. Thecontinuous operation of the system may be directly observed through thevisualization window to determine whether a flow bypass is required orwhether the detection process is complete.

In the illustrated embodiments the magnetic beads are described may befunctionalized with a fluorescent marker. In these embodiments theapertured plate 18 is generally opaque so fluorescence signals comingfrom the unbound beads M_(B) in the bottom chamber 16 b will not bedetected through the viewing panel 20. However, in cases some free beadsmay remain on the surface 51 and produce fluorescence signals that canconfuse the visualization. In these cases the beads can befunctionalized only with ligands and not with fluorescent markers. Afterthe sample has been fully processed and the target entities captured onthe apertured plate 18, fluorescent-tagged ligands may be introducedseparately into the reservoir to bind to the target entities directly.The target entities can then be easily observed through thevisualization panel 20. With this modified approach, in some cases theplate 18 can be devoid of any micro-apertures since the target entitieson the plate surface can be readily differentiated from unbound magneticbeads by means of fluorescence.

In the detection process, the lower chamber 16 b can be flooded with aminute amount of buffer or blood, after which here is no fluid flowthrough the lower chamber until the detection is complete. Fluiddiffusion through the openings 50 between the upper and lower chambers16 a, 16 b will be minimal since this microfluidic detection system 10is not based on a pressure-driven flow.

CTCs from blood samples of patients of various cancers, including butnot limited to prostate, ovarian breast, colon, renal and lung cancers,can be detected since many cancer cells express certain molecules orantigens on their surfaces which can be targeted with variousrecognition elements, including but not limited to antibodies (e.g.,EpCAM), aptamers, low molecular weight ligands (e.g., folate and DUPA)and peptides. The beads can be functionalized as previously described(e.g., DUPA for prostate cancer cells, and folate end EpCAM for ovarian,breast, colon, renal and lung cancer cells) and then incubated and mixedwith the sample fluid for 20-30 minutes. This incubation time may belonger or shorter depending on the number of beads used, sample volumeand the number of target entities sought. For instance, seeding thesample with a larger number of beads increases the chance that a targetcell will “find” a magnetic bead and bind. If there are multiplesamples, incubation of all samples can be carried out simultaneously.The analysis of the sample fluid by the present detection system, andthe sequence of sample fluid and buffer flows can be carried out asdescribed herein. Aliquots of the captured cancer cells can be stainedwith additional recognition elements, such as antibodies to cytokeratinsand EpCAM to assure that the cells retained by the detector componentare indeed cancer cells. Furthermore, a preliminary indication ofwhether CTC numbers correlate with the stage of the disease of thesample donor can be ascertained. While the disclosed system is notlimited to a particular type of cancer, prostate and ovarian cancercells are especially mentioned herein as models for the system becauseboth diseases can have vague symptoms resulting in confusingbiomolecular tests, and both can benefit from a reliable, fast andsensitive CTC test. For example prostate cancer can have symptomssimilar to benign prostatic hyperplasia. PSA biomarker tests can beconfusing due to both false positives and false negatives. Similarly,ovarian cancer can have vague symptoms and may be detected as late asstage III or IV.

In comparison with a number of studies which successfully used magneticbeads to manually separate a wide variety of cells (from pathogens to Tcells to CTCs) from complex samples, the approach according to thepresent disclosure advantageously offers separation-and-detection,faster analysis and the ability to harvest the captured cells and makethem available for other types of analyses, including but not limited togenetic analysis. Furthermore, in comparison with giant magnetoresistive (GMR) or spin-valve sensors which are known to a person ofordinary skill in the art (made of multiple nanolayers withprecisely-controlled thicknesses) that can detect magnetic beads, thepresent approach advantageously is more robust, easy to construct anduse, and offers much higher throughput. The system according to thepresent disclosure also offers a significant advantage over size-basedcell entrapment assays which force the cells through micron-sizedcavities by fluidic pressure. These assays can suffer from clogging ofthe cavities (since all entities in the sample are forced to passthrough the cavities), or trapping of other entities similar in size totarget entities, or complete passage and hence loss of target entitiesthrough the cavity.

The source S may include target entities that have already been bound torecognition elements, as well as magnetic beads. Alternatively thesource may initially contain a sample fluid, such as a whole bloodspecimen, to which magnetic beads are added and allowed to incubate.Since the target entities are rare or at a low concentration, it isdesirable to seed the specimen with millions of functionalized magneticbeads. For instance, in one approach 100 million beads are provided foreach mL of whole blood specimen. A twenty minute incubation time hasbeen found to be sufficient to bind rare CTCs. The system 10 may beprimed as described above during the incubation period since the samplesource S is not involved in the fluid flow during this step. Once theincubation period is complete the flow protocol for detecting the targetentities described above may be implemented. Alternatively, as describedabove, the sample provided may be a blood sampled processed by acombination of commercial cell preparation tubes and centrifugation inorder to discard red blood cells which are usually not sought during aCTC detection. Alternatively, the sample may be a processed blood samplewherein the red blood cells have been lysed using a red blood cell lysisbuffer.

It is contemplated that the system 10 may be modified for incorporationinto a dialysis or dialysis-type system. In this instance,functionalized magnetic beads may be injected into the patient's bloodstream prior to dialysis. Rather than flowing into a collection vesselCC the blood flowing through the detector component 12 or detectorcomponents 12 i is returned to the dialysis. A magnetic collectionelement may be incorporated at the system output to capture all unboundmagnetic beads and bound cells that have not been collected within thedetector component(s).

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Therefore,above disclosure is not to be limited to the specific embodimentsillustrated and described above. The description as presented and asthey may be amended, encompass variations, alternatives, modifications,improvements, equivalents, and substantial equivalents of theembodiments and teachings disclosed herein, including those that arepresently unforeseen or unappreciated, and that, for example, may arisefrom applicants/patentees and others.

For instance, in the exemplary embodiments particular biological cells,such as CTCs, are described as being captured by the detector component12 of the microfluidic detection system 10. Other entities, includingmolecules, particles or similar bodies, in the micrometer (μm) sizerange may also be detected and captured by the system disclosed herein,provided the particles or bodies can bind to one or more recognitionelements, such as the magnetic beads described herein.

Furthermore, the apertured plate 18 is depicted as being generallyparallel with the lower half 15 b of the detector component base 15.Alternatively the plate may be angled toward the outlet channel 23 sothat captured target entities tend to accumulate from the outlet end andfill in toward the inlet end. An angled plate may also facilitatepassage of the unbound magnetic beads into the lower chamber 16 b byinducing a translation along the surface 51 of the plate. Moreover, theplate 18 is shown as generally planar, although other configurations arecontemplated that facilitate capturing target entities and passage ofunbound beads.

The magnet 32 is described herein as a permanent magnet with theapplication of the magnetic field controlled by moving the magnet. Themagnetic field may be manipulated by an adjustable shield disposedbetween the magnet and the reservoir 16. The shield may be used tocompletely block the magnetic field or to reduce the field as necessary.Alternatively the magnet may be an electromagnet that can be controlledby the controller 45 to activate or de-active the magnetic field oradjust the strength of the field. The controller may also modulate themagnetic field during a detection cycle to facilitate capturing thetarget entities and drawing the unbound magnetic beads into the lowerchamber.

In a further alternative the magnet 32 may include multiple magnetsarranged in a predetermined pattern to facilitate counting cellscaptured on the apertured plate 18. Thus, in one embodiment severalmagnets may be arranged in parallel strips so that the captured cellsappear in several lines.

In the fluid flow protocols described above, the target entities areflushed from the detector component 12 in one step. Alternatively thecells may be retained on the apertured plate and the plate itselfremoved from the detector component. In this alternative the magneticcan remain in position as the upper body half 15 a is removed to provideaccess to the apertured plate. The lower body half 15 b may betransported with the apertured plate and magnet intact and mated withanother body half for further procedures.

The detector component 70 shown in FIG. 7 may be implemented in a dualdetection protocol in which two different target entities may bedetected in a single common fluid sample. For instance, the presence ofcertain target cells may be interpreted in view of the presence, orabsence, of certain target molecules, such as proteins, DNA, circulatingDNA, RNA, peptides, small molecules and antigens like the prostatespecific antigen (PSA). Micro-beads may be functionalized to bind withthese target molecules in the same manner described above for thegeneric target entities or target cells. In the dual detection protocol,target cells bound to the magnetic micro-beads are retained on theapertured plate 85, as described above. The un-bound micro-beads alsopass through the apertures in the plate into the second chamber 79, alsoas described above. However, in the dual detection protocol, targetmolecules bound to micro-beads also pass through the apertures in theplate, since the apertures are sized to prevent passage of only cellsand not molecules (or micro-beads).

The target molecules are thus present in the lower second chamber 79 andmore particularly are held against the detection surface 88 by themagnet 82. In one aspect of this dual detection protocol, the detectionsurface 88 is functionalized with certain secondary molecules or ligandsthat can recognize the target molecules captured by the beads. Thetarget molecules bound to the beads and to the secondary ligands thusform a sandwich assay on the surface 88. This assay may be visualized asshown in FIG. 8 in which a laser 90 directs a collimated beam through aprism 91 that is flush with the lower body half 86, with the reflectedbeam passing to a detector 92. In this approach, the surface 88 can becovered with a metallic layer that enables the use of surface plasmonresonance for interrogation of the surface. The surface 88 may befurther modified to incorporate ordered nano-hole arrays with hole sizesof only a few hundred nanometers or less. The nano-hole arrays canenhance the resonance and affect the absorption characteristics of thetarget entities.

It is further contemplated that the surface 88 at the base of thechamber 76 may be functionalized with different secondary ligands ormolecules configured to recognize and capture different targetmolecules. Thus, the surface 88 may be divided into multiple segments orregions, each being dedicated and functionalized to capture a specifictype of molecule. In one embodiment, this multiple functionalization maybe accomplished by a PDMS stamp or other arraying or injectiontechnique. The surface 88 may be functionalized in the form ofalternating lines to create a diffraction grating template, as shown inFIG. 9. The binding of the functionalized beads and target molecules canthen form a diffraction grating so that upon illumination by the lightsource 95 a diffraction pattern appears that allows qualitative andquantitative detection of captured molecules. Multiple diffractiongratings may be provided for multiple different target molecules.

In one process, the target cells captured on the apertured plate 85 canbe analyzed, such as by bright-field or fluorescent microscopy throughthe viewing window 80. It is understood that the second magnet 84 ispreferably moved away from the device 70 to permit access to the viewingwindow. Moreover, it is understood that the first magnet 82 remains inposition to hold the captured cells on the plate 85 during analysis.Once the target cells on the plate have been analyzed the captured cellsand associated micro-beads can be flushed and retrieved at the outlet 78in the manner described above. Once the upper chamber 76 has beenflushed, a buffer solution can be flowed through the lower chamber 79 towash away the unbound beads, leaving behind the bead-captured targetmolecules that are held by the secondary molecules or ligands on thesurface 88. The captured molecules can then be analyzed by optical orfluorescence analysis from beneath the body portion 86. It can beappreciated that the body portion may be formed of a transparentmaterial or may incorporate a viewing window aligned with the bottomchamber 79. The analysis of the captured molecules may include countingthe number of beads, which is a direct count of the number of targetmolecules in the sample. The density of the beads on the surface can beevaluated as proportional to the concentration of the target moleculesin the sample. As an alternative, the body portion 86 may be removed foranalysis and measurement separate from the device. In a furtheralternative, the surface 88 may be non-functionalized with the onlyretention occurring due the magnet 82.

The detection components described herein provide an efficient andaccurate avenue for analyzing a wide range of fluid samples, human bodyfluids or environmental fluids, for the detection of cellular andmolecular targets. Multiple cellular targets in a given sample can bedetected by providing magnetic beads conjugated with different ligandsadapted to capture different cells. The ligands may be conjugated withdifferent colors of fluorescent dyes for easy and immediate recognitionusing known microscopy techniques. As another or additional alternative,magnetic beads of different sizes can be conjugated with differentligands. When the target cells are captured on the apertured plate, theparticular target cells can be differentiated by the size of the bead towhich the cell is bound.

Differently sized beads can lead to a modified detection component withtwo levels of detection surface. One such modified detection component100 is depicted in FIG. 10 in which the body 102 defines three chambers104, 105 and 106. The upper chamber 104 is in communication with theinlet 110 and outlet 112. The intermediate chamber 105 is separated fromthe upper chamber 104 by a first apertured plate 125 and from the lowerchamber 106 by a second apertured plate 127. The intermediate chamber105 is in fluid communication with an inlet 114 and an outlet 116, whilethe lower chamber 106 is in communication with inlet 118 and outlet 120.The inlets and outlets function like the inlets and outlets of theprior-described detection components, namely to flow a sample fluidthrough the component, and flow a fluid through the chambers to flushthe bound target entities and un-bound entities.

The first and second apertured plates 125, 127 may be configured likethe apertured plates discussed above. However, the apertures aredifferently sized between the two plates to correspond to differentlysized functionalized beads. In particular, the first plate 125 caninclude apertures that are sized to prevent passage of a largerfunctionalized bead and allow passage of a smaller bead to the secondplate 127. The second plate can then operate like the plate 85 of thecomponent 70 discussed above. The first plate 125 effectively captures afirst target entity that binds to the specifically functionalized largerbead while the second plate 127 captures a second target entity thatbinds to a differently functionalized smaller bead. The captured targetentities can then be analyzed as described above.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same should be considered asillustrative and not restrictive in character. It is understood thatonly the preferred embodiments have been presented and that all changes,modifications and further applications that come within the spirit ofthe invention are desired to be protected.

What is claimed is:
 1. A method for detecting at least two differenttypes of target entities in a fluid sample, the method comprising:mixing the fluid sample with a plurality of first magnetic beads and aplurality of second magnetic beads to form a fluid sample mixture,wherein the first magnetic beads bind to first target entities and thesecond magnetic beads bind to second target entities, and wherein eachfirst target entity has a smallest effective dimension greater than alargest effective dimension of each second target entity and greaterthan a largest effective dimension of each of said first and secondmagnetic beads; flowing the fluid sample mixture through a detectorcomponent, wherein the detector component comprises a body defining areservoir; a plate arranged within the reservoir to separate thereservoir into a first chamber and a second chamber, wherein the platedefines a plurality of openings therethrough with each opening having aneffective dimension that is larger than the largest effective dimensionof each of the first and second magnetic beads and of the second targetentities and smaller than the smallest effective dimension of the firsttarget entities; and applying a first magnetic force sufficient (i) todraw at least some of the first and second magnetic beads not bound toone of the first or second target entities through the openings in theplate from the first chamber into the second chamber, (ii) to draw atleast some of the second target entities bound to the second magneticbeads through the openings in the plate from the first chamber into thesecond chamber, and (iii) to attract and hold at least some of the firsttarget entities bound to the first magnetic beads against a surface ofthe plate within the first chamber of the reservoir, such that the firsttarget entities are not dislodged from the surface of the plate by thefluid sample mixture flowing through the first chamber.
 2. The method ofclaim 1, further comprising visualizing the plate surface to determine apresence and/or quantity of the first target entities.
 3. The method ofclaim 1, further comprising visualizing the second chamber to determinea presence and/or quantity of the second target entities.
 4. The methodof claim 1, further comprising: after the first target entities havebeen held to the plate surface, modifying the first magnetic force to alevel sufficient to permit dislodgment of the first target entities fromthe plate surface; and flowing a non-reactive liquid through the firstchamber to dislodge the first target entities.
 5. The method of claim 4,wherein the step of modifying the first magnetic force includes applyinga second magnetic force to pull the first target entities from the platesurface.
 6. The method of claim 1, wherein the step of applying a firstmagnetic force includes modulating the first magnetic force whilecontinuously flowing the fluid sample mixture through the detector. 7.The method of claim 6, wherein the step of modulating the first magneticforce includes applying a second magnetic force to modulate the magneticfield applied to the first and second magnetic beads.
 8. The method ofclaim 1, wherein the first target entities are cells and the secondtarget entities are molecules.
 9. The method of claim 1, wherein thefluid sample mixture is flowed through the first chamber at a flow rateof at least about 1.0 milliliters (mL) per minute.
 10. The method ofclaim 9, wherein the fluid sample mixture is flowed through the firstchamber at a flow rate of between about 1.0 mL/minute and 3.0 mL/minute.11. The method of claim 1, wherein the fluid sample mixture is flowedthrough the first chamber at a linear flow velocity of between about 0.7mm/sec to about 3.0 mm/sec.
 12. The method of claim 8, wherein the cellsare circulating tumor cells (CTC).
 13. The method of claim 8, whereinthe cells are prostate cancer cells and the molecules are prostatespecific antigen (PSA).
 14. The method of claim 1, further comprisingcollecting first target entities from the first chamber, collectingsecond target entities from the second chamber, and then analyzing,processing, or both analyzing and processing the first target entities,the second target entities, or both the first and the second targetentities.
 15. The method of claim 14, wherein the first and secondtarget entities are separately analyzed from the same fluid samplemixture.
 16. The method of claim 1, further comprising recirculating atleast a portion of the fluid sample mixture from an outlet of the firstchamber through a recirculation path to an inlet of the first chamber.17. The method of claim 1, wherein each of the first magnetic beads andthe second magnetic beads has a largest effective dimension of about 100nm to about 5.0 microns.
 18. The method of claim 1, wherein each of theopenings in the plate has an effective dimension of about 3.0 to about5.0 microns.
 19. The method of claim 1, wherein a detection surface inthe second chamber is functionalized to bind specifically to the secondtarget entities.
 20. The method of claim 1, wherein the first magneticbeads are functionalized to bind specifically to the first targetentities and the second magnetic beads are functionalized to bindspecifically to the second target entities.
 21. The method of claim 1,wherein the first magnetic beads and the second magnetic beads are thesame type of bead and all magnetic beads bind to both the first and thesecond target entities.